Structure-activity relationship of sulfonyl piperazine LpxH inhibitors analyzed by an LpxE-coupled malachite green assay

Article abstract of DOI:10.1021/acsinfecdis.8b00364

The UDP-2,3-diacylglucosamine pyrophosphatase LpxH in the Raetz pathway of lipid A biosynthesis is an essential enzyme in the vast majority of Gram-negative pathogens and an excellent novel antibiotic target. The ³²P-radioautographic thin-layer chromatography assay has been widely used for analysis of LpxH activity, but it is inconvenient for evaluation of a large number of LpxH inhibitors over an extended time period. Here, we report a coupled, nonradioactive LpxH assay that utilizes the recently discovered Aquifex aeolicus lipid A 1-phosphatase LpxE for quantitative removal of the 1-phosphate from lipid X, the product of the LpxH catalysis; the released inorganic phosphate is subsequently quantified by the colorimetric malachite green assay, allowing the monitoring of the LpxH catalysis. Using such a coupled enzymatic assay, we report the biochemical characterization of a series of sulfonyl piperazine LpxH inhibitors. Our analysis establishes a preliminary structure-activity relationship for this class of compounds and reveals a pharmacophore of two aromatic rings, two hydrophobic groups, and one hydrogen-bond acceptor. We expect that our findings will facilitate the development of more effective LpxH inhibitors as potential antibacterial agents.

Full text of DOI:10.1021/acsinfecdis.8b00364

Article
pubs.acs.org/journal/aidcbc
Cite This: ACS Infect. Dis. XXXX, XXX, XXX−XXX
Structure−Activity Relationship of Sulfonyl Piperazine LpxH
Inhibitors Analyzed by an LpxE-Coupled Malachite Green Assay
Minhee Lee,† Jinshi Zhao,‡ Seung-Hwa Kwak,† Jae Cho,‡ Myungju Lee,§ Robert A. Gillespie,‡
Do-Yeon Kwon,† Hyunji Lee,† Hyun-Ju Park,∥ Qinglin Wu,‡ Pei Zhou,*,‡ and Jiyong Hong*,†
†Department of Chemistry, Duke University, 124 Science Drive, Box 90346, Durham, North Carolina 27708, United States
‡Department of Biochemistry, Duke University Medical Center, Box 3711, Durham, North Carolina 27710, United States
§College of Pharmacy, Ewha Womans University, 52, Ewhayeodae-gil, Seodaemun-gu, Seoul 03760, Republic of Korea
∥School of Pharmacy, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon, Gyeonggi-do 16419, Republic of Korea
S
* Supporting Information
ABSTRACT: The UDP-2,3-diacylglucosamine pyrophosphatase LpxH in
the Raetz pathway of lipid A biosynthesis is an essential enzyme in the vast
majority of Gram-negative pathogens and an excellent novel antibiotic
target. The 32P-radioautographic thin-layer chromatography assay has been
widely used for analysis of LpxH activity, but it is inconvenient for
evaluation of a large number of LpxH inhibitors over an extended time
period. Here, we report a coupled, nonradioactive LpxH assay that utilizes
the recently discovered Aquifex aeolicus lipid A 1-phosphatase LpxE for
quantitative removal of the 1-phosphate from lipid X, the product of the
LpxH catalysis; the released inorganic phosphate is subsequently quantified
by the colorimetric malachite green assay, allowing the monitoring of the
LpxH catalysis. Using such a coupled enzymatic assay, we report the
biochemical characterization of a series of sulfonyl piperazine LpxH
inhibitors. Our analysis establishes a preliminary structure−activity relationship for this class of compounds and reveals a
pharmacophore of two aromatic rings, two hydrophobic groups, and one hydrogen-bond acceptor. We expect that our findings
will facilitate the development of more effective LpxH inhibitors as potential antibacterial agents.
KEYWORDS: LpxH, LpxE, lipid A, Gram-negative bacteria, novel antibiotics, enzyme-coupled assay
is carried out by three functional orthologs that do not coexist:
he emergence of multi-drug-resistant nosocomial Gram-
Tnegative pathogens poses a serious threat to public health LpxH in β- and γ-proteobacteria,7 LpxI in α-proteobacteria,8
and LpxG in Chlamydiae (Figure 1).9 Among these three
and highlights the urgent need for novel antibiotics to
overcome established resistance mechanisms.1,2 The outer
enzymes, LpxH is most widespread, functioning in the majority
(∼70%) of Gram-negative bacteria and in all of the WHO-
listed priority Gram-negative pathogens,1 rendering LpxH an
excellent antibiotic target.
membrane of Gram-negative bacteria consists of an asym-
metric bilayer, with the inner leaflet enriched of phospholipids
and the outer leaflet decorated with lipopolysaccharide (LPS)
or lipooligosaccharide (LOS). The membrane anchor of LPS
and LOS is a unique saccharolipid, known as lipid A
(endotoxin), that shields bacteria from the damage of external
detergents and antibiotics and causes Gram-negative septic
shock during bacterial infection.3 Constitutive biosynthesis of
lipid A by the Raetz pathway is required for the viability and
fitness of virtually all Gram-negative bacteria in nature and in
the human host.3−6 As the Raetz pathway has never been
exploited by commercial antibiotics, lipid A biosynthetic
enzymes are excellent novel antibiotic targets.
Lipid A biosynthesis in Escherichia coli is accomplished by
nine enzymes, of which the first six enzymes are essential.3,5
Although the chemical transformation of lipid A biosynthesis is
conserved throughout all Gram-negative organisms, the fourth
step of the pathway, the cleavage of the pyrophosphate group
of UDP-2,3-diacylglucosamine (UDP-DAGn) to form lipid X,
Recently, a small molecule inhibitor containing the sulfonyl
piperazine scaffold (referred to as AZ1 below; chemical
structure shown in Figure 1) was discovered to display
antibiotic activity against efflux-deficient E. coli strains.10 Based
on the analysis of spontaneous resistance mutations, the target
was identified as LpxH. Consistent with this designation,
overexpression of LpxH resulted in a significant elevation of
the minimum inhibitory concentration.10
To exploit LpxH in antibiotic development, a robust activity
assay is required to establish the structure−activity relationship
(SAR) of lead compounds. The previously reported 32P-
autoradiographic thin-layer chromatography (TLC) assay9,11 is
the most sensitive method for evaluation of LpxH activity and
Received: December 17, 2018
© XXXX American Chemical Society
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Figure 1. Lipid A biosynthetic (Raetz) pathway. The conversion of UDP-2,3-diacylglucosamine (UDP-DAGn) to lipid X is catalyzed by LpxH
(colored in pink) in the vast majority of human Gram-negative pathogens or its functional paralogs LpxI and LpxG (both colored in green).
Figure 2. AaLpxE-coupled, malachite green assay for LpxH. (A) AaLpxE, but not the catalytically deficient H149Q mutant, efficiently
dephosphorylates lipid X to yield free inorganic phosphate. (B) AaLpxE-coupled malachite green assay for analyzing LpxH activity. (C) Standard
curve of inorganic phosphate in the LpxH reaction conditions containing detergents and Mn2+. (D) Comparison of the specific LpxH activity
determined by 32P-autoradiographic TLC and AaLpxE-coupled malachite green assays. (E) Dose-dependent inhibition of E. coli LpxH by AZ1
determined by the enzyme-coupled malachite green assay, yielding an IC50 value of 0.147 0.002 μM. Error bars represent standard deviations
from triplicate measurements.
inhibition. However, due to the short half-life of 32P and the
complicated procedure for preparation and purification of the
32P-labeled substrate,9,11 such a radioactive assay is incon-
venient for evaluating a large number of LpxH inhibitors over
an extended period. In order to facilitate the development of
LpxH-targeting antibiotics, here we report the development of
a nonradioactive assay for convenient measurements of LpxH
activity. Furthermore, we present the modular synthesis of a
series of sulfonyl piperazine LpxH inhibitors and the
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establishment of a preliminary SAR and pharmacophore model
for this class of compounds.
RESULTS AND DISCUSSION
■
Development of a Nonradioactive, Colorimetric
Coupled Assay for LpxH Activity. Despite the high
sensitivity of the conventional 32P-autoradiographic TLC
assay that has been used to identify catalytically important
residues and establish the metal dependence of LpxH and its
functional paralog LpxG,9,11 its application to the inhibition
analysis of a large number of compounds over an extended
period is hindered by the limited half-life of the 32P-
radiolabeled substrate and the complexity of the substrate
preparation. To address these challenges, we developed a
nonradioactive, colorimetric assay for evaluating the LpxH
activity and inhibition. This assay utilizes the recent discovery
of the lipid A 1-phosphatase LpxE from Aquifex aeolicus
(AaLpxE).12 We found that in addition to its reported activity
on Kdo2-lipid A, AaLpxE, but not the catalytically inactive
H149Q mutant, efficiently and quantitatively dephosphorylates
lipid X, the product of the LpxH catalysis (Figure 2A). As
LpxH is a Mn2+-dependent hydrolase, whereas AaLpxE is not,
the conversion of UDP-DAGn to lipid X and UMP catalyzed
by LpxH can be quenched by the treatment of EDTA.
Subsequent addition of AaLpxE to the reaction mixture
converts lipid X to DAGn and inorganic phosphate (Figure
2B). The release of the inorganic phosphate is then probed by
the colorimetric malachite green assay through the formation
of a complex between malachite green, molybdate, and free
phosphate to yield color change.
Because our assay includes detergents (e.g., Triton X-100)
and Mn2+, we first investigated whether the malachite green
assay is compatible with such an assay condition. A phosphate
standard curve was generated for concentrations between 0
and 200 μM in the standard enzymatic assay buffer (20 mM
Tris-HCl pH 8.0, 0.5 mg/mL BSA, 0.02% Triton X-100, 1 mM
MnCl2) (Figure 2C). We found that the colorimetric signal is
linear with respect to the added phosphate concentration,
suggesting that the presence of detergents and Mn2+ does not
adversely affect the malachite green assay. Using a substrate
concentration of 100 μM, the specific activity of LpxH
measured by the AaLpxE-coupled malachite green assay (95.7
5.2 μmol/min/mg) is indistinguishable from that obtained
from the 32P-autoradiographic TLC assay (93.5 8.7 μmol/
min/mg) (Figure 2D), suggesting that the coupled assay is
suitable for quantitative measurements of LpxH activity. Using
this coupled malachite green assay, we were able to determine
a dose-dependent inhibition curve of E. coli LpxH by AZ1,
yielding an IC50 value of 0.147 0.002 μM (Figure 2E).
Synthesis of AZ1 Analogues toward E. coli LpxH.
Encouraged by the robustness of the newly developed LpxH
activity assay, we embarked on the synthesis of a series of AZ1
analogues in order to establish a preliminary SAR of AZ1. The
structure of AZ1 is highly modular (Figure 1): it contains a
trifluoromethyl-substituted phenyl ring and an N-acetyl
indoline group that are connected by a central sulfonyl
piperazine linker. We envisioned that a variety of AZ1
analogues could be prepared by coupling readily available N-
phenyl-substituted piperazines to N-acyl indoline sulfonyl
chlorides, as illustrated in Figure 3. We anticipated that a
detailed SAR analysis of AZ1 analogues would help identify
essential chemical motifs and minimal structural requirements
for LpxH inhibition, which will in turn allow chemical
Figure 3. Convergent synthesis of AZ1 analogues.
modifications to enhance potency and specificity, decrease
off-target effects, and improve drug performance.
AZ1 Phenyl Group Analogues. To gain insights into the
importance of the trifluoromethyl group of AZ1 in LpxH
inhibition, we prepared AZ1 phenyl group analogues by
replacing the trifluoromethyl group with various functional
groups, including halogen, alkyl, and carboxylate groups.
Commercially available m-substituted phenyl piperazines
(1a−e) were coupled to the commercially available 1-acetyl-
5-indolinesulfonyl chloride 2 (Scheme 1A). The coupling
reaction proceeded smoothly in the presence of Et3N to afford
3a−e (JH-LPH-06, -09, -24, -26, and -25) in 43−74% yield.
Next, treatment of 3-(tert-butyldimethylsilyloxy)aniline 4a13
with 2,2′-dichlorodiethylamine hydrochloride (5) gave the
desired 1-(3-((tert-butyldimethylsilyl)oxy)phenyl)piperazine
(6a) (Scheme 1B).14 Coupling of 6a and 2 followed by final
TBS deprotection gave the phenol analogue 8a (JH-LPH-10)
in 75% yield. Carboxylate analogues 8b (JH-LPH-11) and 8c
(JH-LPH-12) were prepared from 3-carbomethoxyaniline (4b)
in a similar manner (Scheme 1B).
AZ1 N-Acetyl Indoline Group Analogues. To investigate
the role of the N-acetyl and indoline groups of AZ1 in LpxH
inhibition, we generated a series of AZ1 N-acetyl indoline
group analogues. First, we prepared the N-acetylsulfanil
analogue 11 (JH-LPH-15) by replacing the indoline ring of
AZ1 with aniline, but keeping the N-acetyl group (Scheme
2A). We also replaced the N-acetyl group with extended
carbonyl chains such as 1,3-diketohydroxamic acid 13 (JH-
LPH-16) in order to assess the effect of chain elongation and
potential metal binding by the hydoxamic acid group in 13 on
activity. The synthesis of 13 began with coupling of 9 to
commercially available 4-acetamidobenzenesulfonyl chloride
(10). Then, Ac deprotection of the N-acetylsulfanil analogue
11 (JH-LPH-15) followed by coupling of the resulting aniline
with monoethyl malonate gave the oxoacetate 12. Compound
12 was subsequently hydrolyzed by LiOH to afford the
corresponding carboxylic acid. PyBOP-mediated coupling with
NH2OTBS successfully provided the desired hydroxamic acid
analogue 13 (JH-LPH-16) but in low yield (35%) (Scheme
2A). Attempts to install the hydroxamic acid group using ethyl
chloroformate and NH2OH·HCl were not successful. In a
similar manner, the synthesis of the phenyloxalamide analogue
(17, JH-LPH-17) began with coupling of 14 with methyl
chlorooxoacetate to give the oxoacetate 15 (Scheme 2B). Basic
hydrolysis of 15 by LiOH followed by installation of a
hydroxamic acid group completed the synthesis of 17 (JH-
LPH-17).
To mimic the hydrogen bonding capability of the N-acetyl
group, we coupled 9 with 3-carboxybenezne sulfonyl chloride
(18a) and 3-(carbomethoxy)benzenesulfonyl chloride (18b)
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Scheme 1. Synthesis of AZ1 Phenyl Group Analogues
to provide 19a (JH-LPH-20) and 19b (JH-LPH-21),
respectively (Scheme 2C).
Analysis of SAR of AZ1 Analogues. Among the tested
AZ1 analogues, the m-bromophenyl piperazine analogue (JH-
LPH-06) showed the strongest inhibition of LpxH activity
(Table 1). It inhibited 74% of LpxH activity at 1 μM. Several
other AZ1 analogues (JH-LPH-07, JH-LPH-08, and JH-LPH-
25) also showed a useful level of LpxH inhibitory activity. The
LpxH activity assay data provided several valuable insights into
the SAR.
First, among AZ1 phenyl group analogues, analogues with a
hydrophobic substituent on the trifluoromethyl phenyl ring
(e.g., JH-LPH-06 and JH-LPH-25) were active, but analogues
with a polar functional group, such as the phenol analogue JH-
LPH-10 and the benzoic acid analogue JH-LPH-12, were
inactive. A bulky hydrophobic m-substituent group was
beneficial for the activity (JH-LPH-06 vs JH-LPH-09). These
data indicated the importance of the hydrophobicity and size
of the m-substituent on the phenyl ring in LpxH inhibitory
activity.
Extended N-acyl groups were well tolerated and only slightly
reduced the activity of AZ1 analogues (JH-LPH-06 vs JH-
LPH-07). Interestingly, the sulfanilide analogue (JH-LPH-15)
was active, indicating that the indoline ring might not be
essential to LpxH inhibition. However, the extend sulfanilide
analogues (JH-LPH-16 and JH-LPH-17) were less active than
JH-LPH-15, and the benzoic acid analogues (JH-LPH-20 and
JH-LPH-21) were inactive.
The comparison of AZ1, JH-LPH-18, and JH-LPH-19
revealed that the six-membered piperazine ring is optimal for
the LpxH inhibition by AZ1. Larger ring analogues (JH-LPH-
18 and JH-LPH-19) were less potent than AZ1. The more
flexible or extended piperazine analogues (JH-LPH-4, JH-
LPH-14, and JH-LPH-23) were essentially inactive, suggesting
that both the rigidity of the piperazine linker and the
orientation of the trifluoromethyl-substituted phenyl ring are
crucial to the AZ1 activity. Surprisingly, the amide analogue
JH-LPH-22 was inactive, indicating that the sulfonamide linker
is important for the AZ1 activity either by maintaining the
We also prepared the indoline 5-oxobutanoic acid analogues
22a (JH-LPH-07) and 22b (JH-LPH-08) by modifying both
the trifluoromethyl and N-acetyl groups of AZ1, as illustrated
in Scheme 2D. Coupling of the known sulfonyl chloride 2115
with 1-(3-bromophenyl)piperazine (1a) provided the desired
22a (JH-LPH-07), which was subsequently hydrolyzed to
afford 22b (JH-LPH-08).
AZ1 Sulfonyl Piperazine Linker Analogues. In order to
probe whether sulfonyl piperazine linker of AZ1 provides the
optimal distance between the trifluoromethylphenyl ring and
the N-acetyl indoline group and whether it interacts with
LpxH, we prepared several sulfonamide linker analogues.
First, we prepared two larger core ring analogues 26a (JH-
LPH-18) and 26b (JH-LPH-19) by replacing the piperazine
group of AZ1 with 1,4-diazacycloheptane and 1,5-diazacy-
clooctane, respectively (Scheme 3).16 We expected 26a (JH-
LPH-18) and 26b (JH-LPH-19) would provide insights into
the effect of ring size and conformation of AZ1 analogues on
LpxH inhibition.
Next, in order to evaluate the effect of structural rigidity, we
synthesized the flexible acyclic linker analogue 28 (JH-LPH-
14). The known N1-(3-(trifluoromethyl)phenyl)ethane-1,2-
diamine (27)17 was treated with 1-acetyl-5-indolinesulfonyl
chloride (2) to afford the desired acyclic analogue 28 (JH-
LPH-14) in 80% yield (Scheme 4A). The one-carbon
homologated analogue 30 (JH-LPH-23) was prepared in a
similar manner. To take advantage of the potential binding of
the kojic acid group to the di-Mn2+ cluster of the LpxH active
site, we designed and synthesized the AZ1 kojic acid analogue
36 (JH-LPH-04) starting from the known phosphonate 3118
and aldehyde 3219 (Scheme 4B). To define the role of the
sulfonamide group of AZ1, we used the standard EDC−HOBt
amide coupling condition and prepared the corresponding
amide analogue 38 (JH-LPH-22) (Scheme 4C).
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Scheme 2. Synthesis of AZ1 N-Acetyl Indoline Group Analogues
optimal compound geometry or by direct interacting with
LpxH, or both.
evaluated by scoring both active and inactive ligands. Although
inactive ligands were not involved in model generation, they
were used to eliminate hypotheses that do not distinguish
between active and inactive compounds, which is especially
useful when all active ligands share a common structural
scaffold. Figure 4A shows the pharmacophore with the highest
adjusted scores mapped onto the most active compound AZ1.
The pharmacophore model is represented with AHHRR,
indicating that it has one hydrogen-bond acceptor (A), two
hydrophobic groups (H), and two aromatic rings (R). Two
hydrophobic groups and one hydrogen-bond acceptor are
mapped to the piperazine ring, the indoline ring, and the
carbonyl group, respectively. These molecular features are
shared by all active ligands, whereas inactive analogues,
including JH-LPH-19 that has a single atom difference from
Pharmacophore Model of LpxH Inhibitors. Pharmaco-
phore models have been used successfully for data mining and
the design of small molecule libraries. Statistically significant
pharmacophore studies based on the analysis of active and
inactive analogues have been used to identify essential and
unfavorable steric regions and electronic requirements.20
We generated a pharmacophore model of LpxH inhibitors
by analyzing the SAR and mapping common structural features
of the five most active compounds (>45% inhibition at 1 μM;
AZ1, JH-LPH-06, JH-LPH-07, JH-LPH-08, and JH-LPH-25).
A total of 50 five-point hypotheses were generated for LpxH
inhibitors, respectively, by requiring all active ligands matched
to the generated hypotheses. The initial hypotheses were
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involved in the recognition of the 1-phosphoglucosamine
headgroup and the β-hydroxyl groups of the 2,3-diacyl chains
of the product lipid X; additionally, the trifluoromethyl group
points toward the solvent-accessible open space above the
active site, suggesting that substitution of the trifluoromethyl
group with polar functional groups should be well tolerated.
However, our SAR analysis and pharmacophore model show
that replacement of the hydrophobic trifluoromethyl group
with a polar functional group (e.g., the hydroxyl group in JH-
LPH-10 or the carboxylate group in JH-LPH-12) is
detrimental to the inhibitory activity of AZ1, whereas a
bulky hydrophobic substitution (e.g., the bromo group in JH-
LPH-06 or the phenyl group in JH-LPH-25) is well tolerated.
Furthermore, terminal polar functional groups (such as
carboxylates) of the N-acyl chain on the indoline ring (e.g.,
JH-LPH-07 and JH-LPH-08) are well accepted, and the
carbonyl oxygen of the N-acetyl group on the indoline ring is a
highlighted hydrogen-bond acceptor in our pharmacophore
model, which would likely interact with polar residues in the
active site rather than hydrophobic residues in the acyl chain
chamber of LpxH. Taken together, our SAR analysis and
pharmacophore model reveal several inconsistencies of the
AZ1 docking model proposed by Bohl and co-workers21 and
instead support a model that AZ1 is oriented with the
trifluoromethyl group away from the active site and with the N-
acetyl indoline close to the active site.
Scheme 3. Synthesis of Analogues with Larger Core Ring
Linkers
Scheme 4. Synthesis of AZ1 Analogues with Flexible Linkers
and an Amide Linker
CONCLUSION
■
The first six enzymes in the Raetz pathway of lipid A
biosynthesis are essential enzymes and viable antibiotic targets.
So far, most inhibitor development has focused on LpxC, the
second enzyme of the pathway, due to the early discovery of
lead compounds in the 1990s.22 Inhibition of late-stage lipid A
enzymes such as LpxH may be uniquely advantageous as
accumulation of lipid A precursors such as UDP-DAGn is
toxic, which provides a second mechanism of cell killing in
addition to disruption of lipid A biosynthesis.23,24 In order to
facilitate the discovery of effective LpxH inhibitors, we have
developed a nonradioactive, colorimetric malachite green assay
that utilizes the unexpected ability of the recently discovered
AaLpxE enzyme to quantitatively desphosphorylate lipid X to
yield DAGn and free inorganic phosphate. Using this assay, we
have biochemically characterized a series of LpxH inhibitors
based on the sulfonyl piperazine scaffold of AZ1, established a
preliminary SAR, and identified the pharmacophore of this
series of LpxH inhibitors. These efforts will ultimately
contribute to the design and development of more potent
LpxH inhibitors, as has been demonstrated for LpxC
inhibitors.25,26
EXPERIMENTAL SECTION
■
For the synthesis of AZ1 analogues, see the Supporting
Information for details.
Cloning and Purification of E. coli LpxH. The E. coli
LpxH gene was cloned into a modified pET30 vector (EMD
Millipore) containing an N-terminal His10-SUMO-fusion
protein. The sequence verified plasmid was used to transform
BL21(DE3)STAR competent E. coli cells (ThermoFisher).
Cells were grown in the Luria broth medium at 37 °C until
OD600 reached 0.5, induced with 1 mM isopropyl-β-D-
thiogalactopyranoside (IPTG) for 3 h, and then harvested by
centrifugation.
AZ1, have poor overlaps with the pharmacophore model of
LpxH inhibitors (Figure 4B).
While this study was in progress, Bohl and co-workers
reported a docking model of AZ1 for LpxH.21 In their model,
the trifluoromethyl-substituted phenyl ring is located close to
the active site consisting primarily of hydrophilic residues
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Table 1. Specific Activity of E. coli LpxH in the Presence of AZ1 Analogues
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Table 1. continued
a
Data are mean values of three independent experiments. Errors represent standard deviation. N/A: not applicable. ND: not determined.
All of the purification procedures were carried out at 4 °C.
Cells from 8 L of induced culture were resuspended and lysed
in 120 mL of the lysis buffer containing 20 mM HEPES (pH
8.0) and 200 mM NaCl using French Press. Cell debris were
removed by centrifugation at 10000g for 40 min. To the
supernatant, n-dodecyl-β-D-maltopyranoside (DDM) was
added to reach a final concentration of 1.5% (w/v; 29 mM).
After 2 h of incubation, membranes were removed by
centrifugation at 100000g for 1 h. Supernatant from the
centrifugation was diluted to a final volume of 240 mL with the
lysis buffer and added to a column containing 20 mL of HisPur
Ni-NTA resin (ThermoFisher) pre-equilibrated with 100 mL
of the purification buffer containing 20 mM HEPES (pH 8.0),
200 mM NaCl, and 0.0174% (w/v; 0.34 mM) DDM. The
column was washed with 250 mL of the purification buffer
containing 50 mM imidazole, and the His10-SUMO-LpxH was
eluted with 150 mL of the purification buffer containing 300
mM imidazole. The eluted protein sample was concentrated
and further purified with size-exclusion chromatography
(Superdex 200; GE Healthcare Life Sciences) in the
purification buffer.
32P-Autoradiographic TLC Assay for AaLpxE. The 32P-
radiolabeled and unlabeled lipid X and the wild-type and
H149Q AaLpxE were prepared as previously described.12,27 To
investigate the AaLpxE activity toward lipid X as the substrate,
a 10 μL reaction mixture containing 50 mM Tris-HCl (pH
7.5), 0.05% Triton X-100, 100 μM lipid X, and 500 cpm/μL
32P-lipid X was preincubated at 30 °C, and the reaction was
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Figure 4. Pharmacophore model for LpxH inhibitors (green, hydrophobic groups; orange, aromatic rings; rose, hydrogen-bond acceptor). (A)
Alignment of five most active LpxH inhibitors (AZ1, JH-LPH-06, JH-LPH-07, JH-LPH-08, and JH-LPH-25) to the pharmacophore. (B) Alignment
of an inactive ligand (JH-LPH-19) to the pharmacophore.
initiated by addition of 1 μg/mL purified AaLpxE. The
reactions were quenched by spotting 2 μL reaction mixture on
the TLC plate at the specified time points. The plate was dried
and developed in a solvent system consisting of chloroform,
methanol, acetic acid, and water (25:15:4:4, v/v), followed by
analysis using the Typhoon FLA 7000 PhosphorImager
scanner equipped with ImageQuant software (GE Healthcare).
Enzymatic Assay for LpxH. To examine the compatibility
of the malachite green assay kit (Sigma catalog number
MAK307) with our assay condition, the phosphate standard
provided with the kit was diluted into our assay reaction buffer
containing 20 mM Tris-HCl pH 8.0, 0.5 mg/mL BSA, 0.02%
Triton X-100, and 1 mM MnCl2. The linear colorimetric
response to a range of phosphate concentrations up to 200 μM
was confirmed.
The autoradiographic assay protocol for LpxH was adapted
from the previous report9 with minor modifications, including
the use of our current assay buffer conditions as described
above and the reaction incubation temperature at 37 °C
instead of 30 °C.
A typical assay for LpxH using the coupled malachite green
assay protocol contained the assay reaction buffer (20 mM
Tris-HCl pH 8.0, 0.5 mg/mL BSA, 0.02% Triton X-100, and 1
mM MnCl2) with 100 μM UDP-DAGn and 5% DMSO or
inhibitors. The reaction mixtures were preincubated at 37 °C
for 10 min before LpxH was added with a 5-fold dilution to
start the reaction at 37 °C. At the desired reaction time points,
an aliquot of 20 μL of reaction mixture was removed and
added to a well in 96-well half-area plate containing 5 mM
EDTA (final concentration) to quench the LpxH reaction.
Then purified AaLpxE was added to a final concentration of 5
μg/mL. The plate was incubated at 37 °C for 30 min followed
by addition of formic acid to a final concentration of 3.75 M to
quench the reaction. The malachite green reagent was added
with a 5-fold dilution, and the absorbance at 620 nm was
measured after 30 min incubation at room temperature. The
amount of free inorganic phosphate from hydrolyzed lipid X
was determined from the phosphate standard curve and used
to calculate the specific activity of the SUMO-LpxH fusion
protein.
For determination of IC50, up to 80 μM of AZ1 compound
was added to the assay reaction containing 5% DMSO. Our
preliminary analysis showed that despite the strong inhibition
of LpxH activity by AZ1 at 1 μM, there existed significant
levels of enzymatic activity at elevated compound concen-
trations beyond 10 μM. Therefore, the protocol for IC50
determination was adjusted to include 10% DMSO instead
of 5% DMSO for the dose−response analysis of AZ1 to
mitigate the concern of limited compound solubility. The
increase of DMSO concentration had minimal impact on the
specific activity of LpxH. The IC50 value was extracted from
fitting of the dose−response curve of vi/v0 = 1/(1 + [I]/IC50).
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Pharmacophore Modeling. The pharmacophore model
of LpxH inhibitors was generated based on total number of 22
analogues that were tested for their inhibition effects against
LpxH (Table 1). The molecular structures were sketched and
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̈
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsinfec-
dis.8b00364.
General experimental procedures with spectroscopic and
analytical data (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: peizhou@biochem.duke.edu.
*E-mail: jiyong.hong@duke.edu.
ORCID
Hyunji Lee: 0000-0001-8463-5078
Hyun-Ju Park: 0000-0001-6802-1412
Pei Zhou: 0000-0002-7823-3416
Jiyong Hong: 0000-0002-5253-0949
Author Contributions
M.L. and J.Z. contributed equally; P.Z. and J.H. conceived the
project, designed the overall experimental strategy, and
analyzed and discussed the results; M.L., M.J.L., D.K., and
H.L. synthesized the small molecules used in this study; S.-
H.K. and H.-J.P. performed pharmacophore modeling and
other computational studies for the project; R.A.G. made the
initial observation of the lipid X activity for AaLpxE. Q.W.
purified AaLpxE; J.C. purified LpxH; J.Z., Q.W., and J.C.
purified lipid X and UDP-DAGn. J.Z. developed the AaLpxE-
coupled enzymatic assay and characterized the inhibitory effect
of LpxH inhibitors; P.Z. and J.H. wrote the manuscript with
input from all the authors and held overall responsibility for
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported in part by grants from the National
Institute of General Medical Sciences (GM115355), the
National Institute of Allergy and Infectious Diseases
(AI139216), and the Bridge Fund from Duke University
School of Medicine.
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